Phycobilisome light-harvesting efficiency in natural populations of the marine cyanobacteria Synechococcus increases with depth

Cyanobacteria of the genus Synechococcus play a key role as primary producers and drivers of the global carbon cycle in temperate and tropical oceans. Synechococcus use phycobilisomes as photosynthetic light-harvesting antennas. These contain phycoerythrin, a pigment-protein complex specialized for absorption of blue light, which penetrates deep into open ocean water. As light declines with depth, Synechococcus photo-acclimate by increasing both the density of photosynthetic membranes and the size of the phycobilisomes. This is achieved with the addition of phycoerythrin units, as demonstrated in laboratory studies. In this study, we probed Synechococcus populations in an oligotrophic water column habitat at increasing depths. We observed morphological changes and indications for an increase in phycobilin content with increasing depth, in summer stratified Synechococcus populations. Such an increase in antenna size is expected to come at the expense of decreased energy transfer efficiency through the antenna, since energy has a longer distance to travel. However, using fluorescence lifetime depth profile measurement approach, which is applied here for the first time, we found that light-harvesting quantum efficiency increased with depth in stratified water column. Calculated phycobilisome fluorescence quantum yields were 3.5% at 70 m and 0.7% at 130 m. Under these conditions, where heat dissipation is expected to be constant, lower fluorescence yields correspond to higher photochemical yields. During winter-mixing conditions, Synechococcus present an intermediate state of light harvesting, suggesting an acclimation of cells to the average light regime through the mixing depth (quantum yield of ~2%). Given this photo-acclimation strategy, the primary productivity attributed to marine Synechococcus should be reconsidered.

This is a nice example of the collaboration between oceanography and biophysics bridged by photosynthesis research. In this manuscript, the authors showed that phycobilisome of cyanobacterial cells in deeper water had faster decay of fluorescence, suggesting more efficient energy transfer to reaction center. This is interesting as well as only novel finding of this paper. In that sense, Figure 3 alone is essential. This type of concise paper may not be highly evaluated in some types of journals, but the reviewer considers conciseness itself may not be a problem because the fact that the efficiency of energy transfer can be regulated in response to light environments in open sea is very important from the viewpoint of photosynthesis research as well as of ecology. The following points should be considered to improve the manuscript.
1. Discussion is quite qualitative. Even though this is the first report on the depth-dependency of antenna regulation of cyanobacteria in natural water, there are several reports on this point for cultured cells, including the works of authors themselves. Please compare the values of fluorescence decay, fluorescence yield or yield of photosynthesis with the values in the past studies of cyanobacteria and other photosynthetic organisms and discuss them more quantitatively.
2. For the comparison mentioned above, understanding of the precise experimental condition is essential. The current explanation of the method of Time-Correlated-Single-Photon-Counting refers to the reference 56, but the explanation in the paper is very short and insufficient. The explanation in the reference 19 is more thorough. If the experimental condition is the same, refer to reference 19 and remove reference 56. Furthermore, the fluorescence lifetime must differ at different temperatures. Please specify the temperature during lifetime measurements. And if it is low temperature, please discuss about the physiological relevance of the interpretation. 3. To calculate absolute quantum yield of fluorescence, the intrinsic lifetime of a phycobilisome complex is essential. For this purpose, the authors prepare the complex biochemically, but it is hard to judge if the obtained intrinsic lifetime is artificially disturbed or not. If possible, it would be nice to have some supporting data that assure the intactness of the complex. Alternatively, it may be possible to strengthen the reliability of the obtained value by comparing it with those reported for similar preparations in the past. 4. It is interesting to know the "complete picture of the fate of the absorbed energy" but it requires the precise determination of Fv/Fm as well as that of fluorescence yield. In the case of cyanobacteria, determination of correct Fv/Fm is not easy (see e.g. Photosynthesis Research, 133, 63-73). Since the authors seems to use DCMU for the determination of Fm, state transition may not have much interfered with the obtained results. The contribution of phycobilisome and PSI chlorophyll to Fo, however, must have led to underestimation of Fv/Fm. The possible effect of such problem should be discussed.
Minor points 5. Please distinguish "pigment" and "chromophore". Linear tetrapyrrole in phycobilin should be chromophore, not pigment, since it covalently binds to phycobiliprotein. 6. Please explain why cyanobacteria used inefficient phycobilisome in upper water, if it is possible to make it efficient by "enhanced coupling between pigments in the phycobilisome". Light absorbance may not be limiting step in photosynthesis in upper water, but, even so, smaller antenna with higher efficiency must be advantageous.
7. First line of the last paragraph of the Introduction section: "in in" -> "in" 8. First paragraph of the Results section: Please first spell out "CTD" or explain it. 9. Second paragraph of the Results section: "chlorophyll increased with depth, indicating a higher concentration of photosynthetic units in cells inhabiting deeper water" is based on the fact that "chlorophyll" is estimated as "per cell" when determined by flow cytometry measurements. This may not be obvious for broad readers of this manuscript.
11. Fifth paragraph of the Discussion section: "mechanism that reacts to the radiation regime" -> "mechanism that respond to the radiation regime"?
12. Fifth paragraph of the Discussion section: "possible due to the higher density of phycobilisome rods" -> "possibly due to the higher density of phycobilisome rods"? 13. Figures 1,2,S1: It may be better to connect symbols of the same season with lines. In the present figures, some symbols are overlapped with one another, and depth-dependency is not so clear.
14. Figure 3: Do not use arbitrary axis for temperature (X axis) for different panels. It makes the comparison between the panels difficult.
15. Figure S2: Why the measuring depth is different for different seasons? Please add some explanation in the text.
Reviewer #2 (Remarks to the Author): The manuscript of Kolodny et al. presents novel and interesting data on the photophysiological characteristics of in-situ cyanobacterial (Synechococcus) populations in the ocean. The study builds on previous culture work by the same group (Kolodny et al. 2020 FEBS J), and effectively amounts to an in-situ confirmation of the key result in this previous work, namely that Synechococcus exhibits a decreased fluorescence lifetime within the phycobilisome and hence apparently an associated increased transfer efficiency under decreased light conditions. In general the manuscript is well written and presented and the results are likely to be of interest, although predominantly probably within the field rather than of wider interest. There were some aspects of the work which I would like to see improved before recommending publication. In particular that were a number of areas where more detail is required in the methods. This is particularly important as the TCSPC measurements are still some of the first reported from natural oceanic populations of phytoplankton and, to my knowledge, the first depth profiles. There are also a few places in the results where I think the authors have reported information in error. These and other more minor points are outlined further below.

Specific comments:
Line 41: I don't follow this. The measured lifetimes are shorter at depth, so shouldn't the fluorescence quantum yield be smaller (i.e. see equation line 97).? I think this is probably a typo and the authors have these values the wrong way around? i.e. the fluorescence quantum yield is 18% at 70m and 3.5% at 130m? See also Line 224 (and associated comment below).
Line 50: It is perhaps worth noting that Prochlorococcus (which is numerically the dominant prokaryotic primary producer in many open ocean systems) does not have a phycobilisome. As a broader contextual question, presumably the phenomenon described is specific to the phycobilisome? Line 203: Either 'must travel a longer distance' or 'must travel longer distances' Line 210: 'fit with our previous laboratory study' Line 224: See previous comment. Taking a value of the intrinsic lifetime of around 2.1 I calculate fluorescence quantum yields of 18% and 3.3% at 70 and 130m respectively.
Lines 225-226: It is unfortunate if, as appears, there is no direct measurement of Fv/Fm from the in situ samples? I would be cautious in the use of these culture data in comparison to the in situ measurements. At the very least, all of the potential caveats in this calculation have to be clearly outlined to the reader. For example, nutrient status will likely be different for the insitu population versus the culture and may have an influence.
Line 247: Not sure this statement can be fully defended as prior measurements of Fv/Fm with depth already indicated that photochemical efficiencies increased at lower light intensities? Also, Line 248, do these models actually make explicit assumptions about the photochemical (or transfer) efficiencies?
Line 267: pre-concentration step. Did you measure Fv/Fm before and after pre-concentration in order to establish whether there was any change? If not did you have any other way of establishing whether the pre-concentration step might have influenced the measurements?
Line 269: how were the samples stored in the dark? E.g. what temperature?
Line 283: assume '574/26' is peak and half bandwidth of emission band? Please state this.
Line 287: suggest 'excitation was performed at 490 nm' Line 288: was all the signal between 515-675 nm averaged? As indicated above, I would like to see more example data presented, including emission spectra if available and decay curves (see e.g. 1 The first fatal problem of this paper is the simple experiment design doesn't not support the proposed generalized photosynthetic energy usage model. Only fluorescence lifetime data was collected that is related photosynthesis, and even this lifetime was set for measure phycobilisome lifetime, not the photosynthesis. We thank all three reviewers for their reports, and hereby address all of the reviewer's comments point by point, after changing the manuscript accordingly.
Changes in the manuscript are highlighted.
In particular, we address all the concerns raised by reviewer 3 regarding the experimental design and the use of phycobilisome lifetime to estimate lightharvesting efficiency. To address these comments, we performed additional experiments and revised the text to explain more precisely the arguments presented in the manuscript (see new Supplemental figure 3).

Reviewer comments
Reviewer #1 (Changes in the manuscript following reviewer's #1 comments are highlighted in yellow throughout the manuscript): This is a nice example of the collaboration between oceanography and biophysics bridged by photosynthesis research. In this manuscript, the authors showed that phycobilisome of cyanobacterial cells in deeper water had faster decay of fluorescence, suggesting more efficient energy transfer to reaction center. This is interesting as well as only novel finding of this paper. In that sense, Figure 3 alone is essential. This type of concise paper may not be highly evaluated in some types of journals, but the reviewer considers conciseness itself may not be a problem because the fact that the efficiency of energy transfer can be regulated in response to light environments in open sea is very important from the viewpoint of photosynthesis research as well as of ecology. The following points should be considered to improve the manuscript. Reference 19 was also added as suggested by the reviewer, however it is important to note that the setup used there also includes the description of temperature dependent measurements, which were not performed in the present research. These measurements are experimentally demanding due to the limitations of the cryogenic systems and require at least 24h for each sample, making them irrelevant for samples acquired at a field study, which must be measured simultaneously for comparison.

‫פרופ‬
3. To calculate absolute quantum yield of fluorescence, the intrinsic lifetime of a phycobilisome complex is essential. For this purpose, the authors prepare the complex biochemically, but it is hard to judge if the obtained intrinsic lifetime is artificially disturbed or not. If possible, it would be nice to have some supporting data that assure the intactness of the complex. Alternatively, it may be possible to strengthen the reliability of the obtained value by comparing it with those reported for similar preparations in the past.
Reply: This point was also raised by reviewer 3. Additional information on the subject can be found in the response to his critique. Values for the intrinsic lifetime are available for chlorophyll. However, we couldn't find previous reports for PBS. Following the reviewers' comments, we isolated phycobilisomes and estimated their intrinsic lifetime and their fluorescence yield through intensity measurements performed in an integrating sphere spectrometer in addition to the fluorescence lifetime measurements. Information supporting the intactness of the PBS fraction used for the experiments is added as supplementary information (Fig. S3 A). 4. It is interesting to know the "complete picture of the fate of the absorbed energy" but it requires the precise determination of Fv/Fm as well as that of fluorescence yield. In the case of cyanobacteria, determination of correct Fv/Fm is not easy (see e.g. Photosynthesis Research, 133, 63-73). Since the authors seems to use DCMU for the determination of Fm, state transition may not have much interfered with the obtained results. The contribution of phycobilisome and PSI chlorophyll to Fo, however, must have led to underestimation of Fv/Fm. The possible effect of such problem should be discussed.
Reply: We agree and a discussion on the limitation of Fv/Fm measurements in cyanobacteria was added. This section can be found in lines 258-269.
Reply: The term was corrected all along the manuscript following the reviewer's comment (highlighted in the text).
6. Please explain why cyanobacteria used inefficient phycobilisome in upper water, if it is possible to make it efficient by "enhanced coupling between pigments in the phycobilisome". Light absorbance may not be limiting step in photosynthesis in upper water, but, even so, smaller antenna with higher efficiency must be advantageous.
Reply: The following sentence and reference were added to put PBS energy transfer efficiencies in context: "The plasticity of the Synechococcus is enabled by the position of the PBS antenna in the inter-thylakoid space. However, at the same time, the intermediate chromophore coupling regime determines energy transfer efficiencies that are considered lower than those of thylakoid membrane internal antenna complexes (ref 60 7. First line of the last paragraph of the Introduction section: "in in" -> "in" Reply: Corrected in the re-submitted version and highlighted in the text.
8. First paragraph of the Results section: Please first spell out "CTD" or explain it.
Reply: Corrected in the re-submitted version and highlighted in the text.
9. Second paragraph of the Results section: "chlorophyll increased with depth, indicating a higher concentration of photosynthetic units in cells inhabiting deeper water" is based on the fact that "chlorophyll" is estimated as "per cell" when determined by flow cytometry measurements. This may not be obvious for broad readers of this manuscript.
Reply: Corrected in the re-submitted version and highlighted in the text. Reply: Corrected in the re-submitted version and highlighted in the text.
11. Fifth paragraph of the Discussion section: "mechanism that reacts to the radiation regime" -> "mechanism that respond to the radiation regime"?
Reply: Corrected in the re-submitted version and highlighted in the text.
12. Fifth paragraph of the Discussion section: "possible due to the higher density of phycobilisome rods" -> "possibly due to the higher density of phycobilisome rods"?
Reply: Corrected in the re-submitted version and highlighted in the text. 13. Figures 1,2,S1: It may be better to connect symbols of the same season with lines. In the present figures, some symbols are overlapped with one another, and depth-dependency is not so clear.
Reply: Corrected in the re-submitted version.
14. Figure 3: Do not use arbitrary axis for temperature (X axis) for different panels. It makes the comparison between the panels difficult.
Reply: We tried to follow the reviewer's suggestion, however setting the axis range according to the temperature axis hinders the visibility of the lifetime data. Since the temperature is the secondary factor shown in this figure, while the main factor presented is the lifetime data, we prefer to fix the lifetime axis for easier comparison. To ease the comparison between temperatures, we added labels of temperatures on the lower x-axis for the convenience of the readers.
15. Figure S2: Why the measuring depth is different for different seasons? Please add some explanation in the text.
Reply: The methods section describing the sampling depths was elaborated: "Depths were chosen in order to capture the different states of the water column during summer: the stratified layer, the DCM, and below the DCM. Same depths were followed during winter mixing. An additional depth of 25 m was added during two cruises in October-November, which further characterized the shallower communities. During July and August, samples for flow cytometry were collected from two depths only, that represented stratified shallow 5m and the DCM.". This section is highlighted and can be found in lines 304-309 of the manuscript.
Reviewer #2 (Changes in the manuscript following reviewer's #2 comments are highlighted in green throughout the manuscript): wider interest. There were some aspects of the work which I would like to see improved before recommending publication. In particular that were a number of areas where more detail is required in the methods. This is particularly important as the TCSPC measurements are still some of the first reported from natural oceanic populations of phytoplankton and, to my knowledge, the first depth profiles. There are also a few places in the results where I think the authors have reported information in error. These and other more minor points are outlined further below.
Specific comments: Line 41: I don't follow this. The measured lifetimes are shorter at depth, so shouldn't the fluorescence quantum yield be smaller (i.e. see equation line 97).? I think this is probably a typo and the authors have these values the wrong way around? i.e. the fluorescence quantum yield is 18% at 70m and 3.5% at 130m? See also Line 224 (and associated comment below).
Reply: We thank the reviewer for detecting this important "typo". The numbers were indeed flipped by mistake. It was corrected both in the abstract and in the discussion. This section can be found in lines 41-42, 185 of the manuscript (highlighted in the text).
Line 50: It is perhaps worth noting that Prochlorococcus (which is numerically the dominant prokaryotic primary producer in many open ocean systems) does not have a phycobilisome. As a broader contextual question, presumably the phenomenon described is specific to the phycobilisome?
Reply: Following the reviewer's suggestion this comment was added to the introduction and the difference between the antenna systems of these strains is now elaborated: "Prochlorococcus however, use membrane internal light harvesting systems." This section is highlighted and can be found in line 57 of the manuscript.
Line 57: '..light harvesting in the deeper ocean' Reply: Corrected in the re-submitted version and highlighted in the text.
Line 79: '… when grown under lower light.' Reply: Corrected in the re-submitted version and highlighted in the text.